The field of this invention is immunology, specifically, methods of preparing artificial antigen presenting cells and their application to methods of isolating antigen-specific T cells, methods of modulating the T cell response and methods of treating conditions which would benefit from the modulation of the T cell response, for example, transplantation therapy, autoimmune disorders, allergies, cancers and viral and bacterial infections.
The following description provides a summary of information relevant to the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to the invention.
The immunologic arts have advanced markedly over the past ten years. The complexity of the science explaining aspects of the field is immense. We set forth below in this section a discussion concerning known aspects of various elements involved in immunogenic responses and concepts in the art that are related to the invention disclosed herein.
T Cells
T lymphocytes (i.e., T cells) are part of the immune system, which defends the body against bacterial, viral and protozoal infection, as well as aberrant molecules that contain epitopes recognized as non-self. The recognition of non-self molecules as well as the destruction of infectious agents carrying non-self antigens is a function of T cells. These cells provide for the cell-mediated immune responses of adaptive immunity.
Infecting pathogens are generally accessible to extracellular antibodies found in the blood and the extracellular spaces. However, some infecting agents, and all viruses, replicate inside cells where they are not exposed to, and cannot be detected by, extracellular antibodies. In order for these foreign agents to be accessible to the cell-mediated immune response, the cells harboring such pathogens must either xe2x80x9cexpressxe2x80x9d antigenic motifs of the infecting agents on the surface of the cells or the antigenic motifs must be shed from the cells, e.g. by cell death, to be accessable to and subsequently expressed on the cell membrane of phagocytic antigen presenting cells (xe2x80x9cAPCsxe2x80x9d) that participate in the immune process.
Antigens derived from replicating virus for example, are displayed on the surface of infected cells where they may be recognized by xe2x80x9ccytotoxicxe2x80x9d T cells which may then control the infection by recognizing the viral antigen and killing the cell. The actions of such cytotoxic T cells depends upon direct interaction between the antigenic motif of the infecting agent expressed on the surface of the infected cells and the T cell""s receptors having a specificity for the motif.
Although T cells are important in the control of intracellular infections, some foreign agents evade such control because they replicate only in the vesicles of macrophages; an important example is Mycobacterium tuberculosis, the pathogen that causes tuberculosis. Whereas bacteria entering macrophages are usually destroyed in the lysosomes, which contain a variety of enzymes and bactericidal substances, infectious agents such as M. tuberculosis, survive because the vesicles they occupy cannot fuse with the lysosomes. The immune system provides for fighting such agents by a second type of T cell, known as a T helper cell, which helps to activate macrophages and induce the fusion of lysosomes with the vesicles containing the infecting agents. The helper cells also bring about the stimulation of other immune mechanisms of the phagocyte. T helper cells may further be involved in initiating and/or sustaining the immune system""s release of soluble factors that attract macrophages and other professional APCs to the site of infection.
Additionally, specialized xe2x80x9chelperxe2x80x9d T cells play a central part in the destruction of extracellular pathogens by interacting with B cells. Depending on the type of infection being controlled, participating T helper cells may have an inflammatory or Th1-like phenotype, or a suppressive Th2-like phenotype.
T Cell Receptors
T cell receptors (TCRs) are closely related to antibody molecules in structure and are involved in antigen binding. Variability in the antigen binding site of the TCR is created in a fashion similar to antibodies in that a large capacity for diversity is available. The diversity is found in the CDR3 loops of TCR variable regions which are found in the center of the antigen-binding site of the TCR. The diversity that is obtainable by TCRs for specific antigens is also directly related to an MHC molecule on the APC""s surface to which the antigenic motif is bound and presented to the TCR.
One type of MHC that is involved in presenting processed antigen is class II MHC. The antigen or peptide binding site for a peptide on a class II MHC molecule lies in a cleft between the alpha and beta chain helices of the MHC molecule. In another type of MHC, the class I MHC, the binding site for a peptide lies in a cleft between the two alpha helices of the alpha chain. From the arrangement of highly variable antigens complexing with MHC molecule alleles, it is understandable that the mechanism of TCR recognition involves a combined distribution of variability in the TCR which must correlate with a distribution of variability in the ligand (i.e., antigen/MHC molecule complex). (Garboczi, et al., Nature Vol. 384; 134-41; Ward and Quadri, Curr Op Immunol. Vol. 9:97-106; Garcia, et al., Science, Vol. 279:1166-72).
MHC Molecules
In general, T cell responses to non-self motifs depend on the interactions of the T cells with other cells containing proteins recognized as non-self. In the case of cytotoxic T cells and Th1 cells, non-self proteins (i.e. antigens) are recognized on the surface of the target cell (such as an infected cell). Th2 cells, on the other hand, recognize and interact with antigen presented by professional antigen presenting cells such as dendritic cells, and B cells. Dendritic cells non-specifically internalize antigen while B cells bind and internalize foreign antigens via their surface immunoglobulin. In any case, T cells recognize their targets by detecting non-self antigenic motifs (e.g., peptide fragments derived from for example, a bacterium or virus) that are expressed either on infected cells or other immune cells, e.g. phagocytic APC. The molecules that associate with these peptide or antigen fragments and present them to T cells are membrane glycoproteins encoded by a cluster of genes bearing the cumbersome name xe2x80x9cmajor histocompatibility complexxe2x80x9d (MHC). These glycoproteins were first identified in mice in studies examining the effects on the immune response to transplanted tissues. In humans, the MHC equivalent has been termed HLA for xe2x80x9chuman leukocyte antigenxe2x80x9d. In general, the term MHC is used to describe generally the molecules in the mammalian immune system involved in the presentation of antigenic motifs to T cells. As used specifically in this Letters Patent, MHC means any major histocompatibility complex molecule, either class I or class II, from any mammalian organism including a human, such molecule comprising full-length MHC molecules or sub-units thereof further comprising MHC encoded antigen-presenting glycoproteins having the capacity to bind a peptide representing a fragment of an auto antigen or other non-antigenic or antigenic sequence (e.g., a peptide), said MHC further having an amino acid sequence that is expressed and purified from natural sources, or by any artificial means in prokaryotic or eukaryotic systems having different glycosilations, or of either natural or synthetic origin that contains or comprises a modification of a natural MHC sequence.
The actions of T cells depend on their ability to recognize antigenic motifs on cells (such as cells harboring pathogens or that have internalized pathogen-derived products). T cells recognize peptide fragments (e.g., pathogen-derived proteins) in the form of complexes between such peptides and MHC molecules that are expressed on the surface of xe2x80x9cantigen presenting cellsxe2x80x9d.
The two types of MHC molecules, i.e., MHC class I and MHC class II deliver peptides from different sources (class I being intracellular, and class II being extracellular) to the surface of the infected cell. The two classes of MHC molecules vary with respect to the length of peptides that they are able to present. The binding pocket of the MHC class I molecules is blocked at either end, thereby imposing severe restrictions on the size of peptides it can accommodate (8-10 residues). The binding groove of the MHC class II molecules on the other hand allows peptides to protrude from the ends, and consequently much longer peptides (8-30 residues) can bind. (Rudensky, et al. Nature, Vol. 353:622-27; Miyazaki, et al., Cell, Vol. 84:531-41; Zhong, et al., J Exp. Med., Vol. 284:2061-66).
Antigen Processing
Peptides bound to MHC class I molecules are recognized by CD8+ T cells (cytotoxic T cells), and those bound to MHC class II molecules are recognized by CD4+ T cells (helper T cells). Two functional subsets of T cells are thereby activated to initiate the destruction of antigenic motifs, and thereby the source (e.g. a pathogen) which may reside in different cellular compartments. CD4+ T cells may also help to activate B cells that have internalized specific antigen, and in turn give rise to the stimulation of antibody production against the antigenic motifs of the extracellular pathogens.
Infectious or antigenic agents can reside in either of two distinct intracellular compartments. Viruses and certain bacteria replicate in the cytosol or in the contiguous nuclear compartment, while many pathogenic bacteria and some eukaryotic parasites replicate in the endosomes and lysosomes that form part of the vesicular system. The immune system has different strategies for eliminating such agents from these two sites. Cells containing viruses or bacteria located in the cytosol are eliminated by cytotoxic T cells which express the cell-surface molecule CD8. The function of CD8 T cells is to kill infected cells.
Immunogenic agents located in the vesicular compartments of cells (which may or may not have been involved in the internalization of extracellular matter) are detected by a different class of T cell, distinguished by surface expression of the molecule CD4. CD4 T cells are specialized to activate/modulate other cells and fall into two functional classes: Th1 cells which activate various immune competant cells to have the intravesicular non-self antigenic agents they harbor destroyed, and Th2 cells which help to activate B cells to, among other things, make antibody against such foreign agents.
To produce an appropriate response to infectious microorganisms, T cells need to be able to distinguish between self and foreign or non-self material coming from the different processing pathways. This is achieved through delivery of peptides to the cell surface from each of these intracellular compartments by the different classes of MHC molecules. As noted above, MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where the antigen (i.e., non-self recognized peptide) is expressed in association with the MHC molecules (antigen:MHC complex) and is recognized by CD8 T cells. Likewise, MHC class II molecules deliver the non-self peptides originating from extracellular sources to the cell surface, where they are recognized by CD4 T cells.
Antigen Presenting Cells
When naive T cells encounter for the first time a specific antigen on the surface of an antigen-presenting cell (APC), they are activated to proliferate and differentiate into cells capable of contributing to the removal of the antigen and its source (e.g. an infecting pathogen). The APCs are specialized in that they express surface molecules that synergize with a specific antigen in the activation of naive T cells. APCs become concentrated in the peripheral lymphoid organs, to which they migrate after trapping antigen while circulating in the periphery. APCs present peptide fragments or antigenic motifs to recirculating naxc3xafve T cells. Arguably, the most important APCs are dendritic cells whose known function includes the presentation of antigen to Macrophages and are important in phagocytosis of cells that provide a first line of defense against infecting agents. APCs are also known to be activated by armed effector T cells. B cells also serve as APCs under some circumstances.
One of the features of APCs is the expression of co-stimulatory molecules including B7-1 and B7-2 molecules. Naxc3xafve T cells will respond to an antigenic motif only when the same APC presents to the T cell both the specific motif recognized by the TCR and a B7 molecule which is recognized by CD28 or CTLA-4, the receptors for B7 existing on the T cell surface. (Anderson, et al., J. Immunol., Vol. 159:4:1669-75). The activation of T cells by APCs leads to proliferation of the activated T cells and to the differentiation of their progeny into armed effector T cells. The proliferation and differentiation of T cells depends on the production of cytokines (such as the T cell growth factor, IL-2) and their binding to high-affinity receptors on the activated T cell. T cells whose TCRs are bound to antigens in the absence of co-stimulatory molecules fail to make cytokines and instead become anergic. This dual requirement for both receptor/antigenic interaction and co-stimulation helps to further mediate naxc3xafve T cell response.
Proliferating T cells develop into armed effector T cells, the critical event in most adaptive immune response. Once an expanded clone of T cells achieves effector function, the T cell clone progeny can act on any target cell that displays or expresses a specific antigen on its surface. Effector T cells can mediate a variety of functions. The killing of infected cells by CD8 cytotoxic T cells and the activation of professional APC by Th1 cells together make up cell-mediated immunity. The activation of B cells by both Th2 and Th1cells help to produce different types of antibodies, thus driving the humoral immune response. (Kirberg, et al., J. Exp. Med., Vol. 186:8:1269-75).
T Cell Activation
T cells generally become sensitized to antigens by becoming trapped in lymphoid organs as the T cells drain into lymph nodes through which they circulate. Antigens introduced directly into the bloodstream, or that reach the bloodstream from an infected lymph node, are picked up by APCs in the spleen for example where lymphoid cell sensitization occurs in the splenic white pulp. The trapping of antigen by APCs that migrate to these lymphoid tissues combined with the continuous recirculation of T cells through the tissues ensures that rare antigen-specific T cells will encounter their specific antigen being presented by an APC.
The recirculation of naxc3xafve T cells through the lymphoid organs is orchestrated by adhesive interactions between lymphocytes and endothelial cells. Naxc3xafve T cells enter the lymphoid organs through a process which is thought to occur in a number of steps. The first step in this process is mediated by selectins expressed on the T cell. For example, L-selectin on naxc3xafve T cells binds to sulfated carbohydrates on the vascular addressins GlyCAM-1 and CD34. CD34 is expressed on endothelial cells in many tissues but is properly glycosylated for L-selectin binding only on the high endothelial venule cells of lymph nodes. L-selectin binding promotes a rolling interaction, which is critical to the selectivity of naxc3xafve lymphocyte homing. Although this interaction is too weak to promote extravasation, it is essential for the initiation of the stronger interactions that then follow between the T cell and the high endothelium, which are mediated by molecules with a relatively broad tissue distribution. (Finger, et al., Nature, Vol. 379:266-9).
Stimulation by locally bound chemokines activates the adhesion molecule LFA-1 on the T cell, increasing its affinity for ICAM-2, which is expressed constitutively on all endothelial cells, and ICAM-1, which, in the absence of inflammation, is expressed only on the high endothelial venule cells of peripheral lymphoid tissues. The binding of LFA-1 to its ligands, ICAM-1 and ICAM-2 plays a major role in T cell adhesion to and migration through the wall of the blood vessel into the lymph nodes. Bachmann et al., Immunity, Vol. 7:549-57).
The high endothelial venules are located in the lymph nodes. This area is inhabited by dendritic cells, which have recently migrated from the periphery. The migrating T cells scan the surface of these APCs for specific antigen:MHC complexes. If they do not recognize antigen presented by these cells, they eventually leave the node via an efferent lymphatic vessel, which returns them to the blood so that they can recirculate through other lymph nodes. Rarely, a naxc3xafve T cell recognizes its specific antigen:MHC complex on the surface of an APC, which then signals the activation of LFA-1, causing the T cell to adhere strongly to the APC. Binding to the antigen:MHC complex also activates the cell to proliferate and differentiate, resulting in the production of armed, antigen-specific T cells. The number of T cells that interact with each APC in lymph nodes is very high, as can be seen by the rapid trapping of antigen-specific T cells in a single lymph node containing antigen.
Identification and Isolation of Antigen-Specific T Cells
As noted above, T cells represent a major component of the body""s immune defenses against bacterial, viral and protozoal infections, as well as non-self antigenic motifs from other sources. T cells have also been implicated in the rejection of cancerous cells. Autoimmune disorders have also been linked to antigen-specific T cell attack against various parts of the body. One of the major problems hampering the understanding of and intervention on the mechanisms involved in these disorders is the difficulty in identifying T cells specific for the antigen to be studied. Accordingly, it is of great interest to be able to identify antigen-specific T cells. Additionally, it would be of great therapeutic benefit if T cells specific for a particular antigen could be (i) enriched and then reintroduced in a disease situation, (ii) selectively depleted in the case of an autoimmune disorder, or (iii) modified to alter their functional and/or phenotypic characteristics. Thus, identification and isolation of antigen-specific T cells is an essential requirement in immunology and medicine to understand and modulate immune responses.
Identification of antigen-specific T cell populations is generally accomplished by indirect means in animal models, such as by evaluating membrane markers correlated to activation or maturation of these cells. The majority of these studies were performed in transgenic systems (Ignatowicz, et al., Cell, 84:521-29; Sebzda et al., Science, Vol. 263:1615-18; Jameson et al., Ann. Rev. Immunol., Vol. 13:93-126). Analysis is generally done by means of flow cytometry, where a detector on a machine is capable of identifying cells bound to fluorescent substrates, such as fluoresceinated antibodies. Positively identified cells can be sorted for further use. Quantitation and isolation of antigen-specific T cells is usually accomplished by limiting dilution and cloning techniques. When using sorted cells, these approaches become quite cumbersome and are sometimes inaccurate, since the biological effects of antigen recognition can spread beyond the cells recognizing the antigen. For instance, upon engagement of the specific MHC:antigenic peptide complex, T cells produce cytokines that can affect expression of the same markers of activation in non-specific bystander T cells. Hence, in order to isolate and characterize cells with specificity for a given antigen, alternative procedures, such as T cell cloning, need to be applied. These techniques often require many months of technical procedures before results can be obtained. The rate of success, in particular for human systems, is quite low, and the population selected may not necessarily represent the biologically relevant component of the immune response to a given peptide. The direct interaction of a specific T cell with the antigen:MHC complex would thus be a preferred basis for T cell isolation.
Theory of the Invention
The immunoregulation art has advanced steadily in recent years. The scientific literature contains many studies showing interactions and modulation effects between specific molecules and cell types. However, no discovery has been presented that is able to apply the knowledge that has been gained by the extensive research in the field toward a method or device that can be used in a comprehensive package for carrying out the identification, isolation, and modulation of immunoregulatory cells for the purpose of advancing the ultimate goal of such knowledge, i.e, improved treatment regimens for various states of disease.
We have discovered a platform technology for advancing treatment regimens requiring the immunoregulation of immune cells that centers around the use of an artificial antigen presenting cell (APC). This platform technology may be designed or programmed on demand for use in the treatment of a broad spectrum of specific disease states. Moreover, this system is versatile and applicable to all situations where the isolation, identification, and modulation of T cells is of clinical import. We have recognized the relevance of several types of molecular entities to the stimulation/activation and modulation response of T cells in their role within the immune system and have incorporated these entities into artificial APCs. We use such artificial APCs to capture and manipulate antigen specific T cells.
Historically, programming and using T cells therapeutically has been hampered by the problem of finding a means by which the cells can be handled for such manipulation and observation of the effectiveness of the manipulation applied. We have solved this problem by adopting the theory that a T cell can best be manipulated by using APC like structures and encorporating into such structures molecules constructed to (1) bind the xe2x80x9cartificialxe2x80x9d APC to specific T cell types, (2) stimulate or modulate only specifically bound T cells for any desired response, and (3) bind the artificial APC to a solid support in situations where anchoring the APC to a specific location is desired.
Prior to our invention, no comprehensive system has been disclosed, nor was it obvious that such a system would function as desired, to achieve a platform that is universally applicable to activating and modulation T cells. As can be seen by the numerous following distinctions, much of the art has centered only on basic research relating to molecules and their association with T cell response.
Distinctions
Kendrick et al., U.S. Pat. No. 5,595,881 (the ""881 disclosure) discuss a method for the detection and isolation of MHC:antigen-restricted T cells which is performed by preparing the MHC:antigen complex, which complex is isolated by using metal chelating technology. The complex is then bound to a planar solid support (i.e., a glass coverslip), followed in turn by combining the immobilized complex with a biological sample so that the MHC:antigen complex may bind to and retain antigen-specific T cells. Determination of the presence of reactive MHC:antigen complexes is carried out by observation of cell proliferation.
The method described in the ""881 disclosure differs from the current invention in a number of substantial structural and functional ways. First, the MHC component of the complexes in the ""881 disclosure are immobilized on a solid support. The MHC component of the current invention is not bound to a solid support but is freely xe2x80x9cfloatingxe2x80x9d within the bilayer of a polysome membrane comprising a phosphotidylcholine and cholesterol component. The difference is substantial in that the MHC:antigen complex of the ""881 disclosure is not able to participate in the migration or concentration of such complexes in xe2x80x9ccappingxe2x80x9d which is important to improved binding and activation of bound T cells. Second, the ""881 method is only directed to the detection of the presence of xe2x80x9cnaturalxe2x80x9d APCs that are specific for pre-selected antigen-specific T cells after such T cells have been isolated. The isolation of antigen-specific T cells is carried out by first performing a series of steps including binding antigen via a metal chelating process to a solid support, capturing on to the antigen MHC the components that are antigen-specific, then isolating the MHC:antigen complexes which are in turn bound to a planar solid support via a linker.
The current invention is also much more versatile. It is not concerned with detecting natural APCs but is instead directed to the isolation and manipulation of antigen-specific T cells. The manipulation of such T cells is carried out for numerous applications such as directly impacting T cell function by modulating the T cell response. The manipulation can be performed in either a column format, with means for supporting the artificial APCs, and/or in free solution via flow cytometry (FACS). The current invention is able to modulate T cell function because the artificial APCs may be designed to specification in that various functional molecules are incorporated into the APC that activate specific T cell responses. For example, in one embodiment of the current invention, known MHC molecules may be incorporated into liposomes along with a labeled antigenic peptide for which such MHC has specificity (e.g., in the case of FACS a biotinylated antigen). The liposome:MHC:biotinylated antigen complex may be used to bind to antigen-specific T cells and the fact of binding can be visualized by FACS followed by the sorting of the bound cells. Thus, no cell proliferation is necessary to identify and isolate antigen-specific T cells.
In addition to the MHC:antigen complex, the xe2x80x9cartificial APCsxe2x80x9d used to capture the antigen-specific T cells include accessory molecules to help stabilize the MHC:antigen:TCR interaction, and may also include functional molecules such as co-stimulatory molecules which in one embodiment may be used to activate T cells, adhesion molecules which may be used to bind cells destined for a certain area of the body, and other accessory or functional molecules such as cytokines or antibodies to cytokine receptors, which are known to have immunomodulatory effects upon T cells. Moreover, the current invention further provides for proper orientation of each of these molecules within the artificial APC membrane by a novel use of an anchoring mechanism comprising GM-1 ganglioside and the xcex2 subunit of cholera toxin. In this aspect, the protein of interest may be connected to the cholera toxin subunit as a fusion protein or by use of a linking moiety. By attaching the cholera toxin subunit to the molecule of interest, the cholera toxin may be bound by the GM-1 that is incorporated into and has affinity for the nonpolar region of the artificial APC membrane.
All of these molecules are incorporated into the liposomes of the artificial APCs in a free floating format. Other molecules may be included that do not influence the modulation of T cell response such as proteins that may be used to anchor the artificial APC to a solid support. Such molecules may also be produced as fusion proteins for proper orientation. As used herein such molecules that are not associated with modulation or T cell binding are termed xe2x80x9cirrelevantxe2x80x9d molecules.
Additionally, a label may be attached to the antigen, the irrelevant molecule, or the liposome component. Moreover, label may also be noncovalently associated within the lipids of the liposome.
The designs of these artificial APCs also allow for optional expansion experimentation of T cell populations responding to the MHC:antigen complexes associated in the cell like liposomes using a solution based (e.g., roller bottle) cell culture. The concept of the current invention represents a substantial and heretofore unrecognized advance in the MHC:antigen complex T cell binding art in that the artificial APC (e.g. the example comprising liposome:MHC:antigen:accessory molecule:functional molecule complex) is not restricted to complexes of MHC:antigen alone or to a planar surface as is the case with much of the prior art. The importance of the structural differences can not be over emphasized. The addition of the accessory molecules, as well as co-stimulatory molecules, and other proteins in proper orientation in the liposomes of the current invention allow for substantially improved binding association and manipulation of T cells which is very important in the identification and stimulation of antigen-specific T cells. This is especially true in solution based FACS analysis where functionality of the antigen-specific T cells can be interpreted directly. For example, prior studies (Watts, T. H. Annals of the New York Academy of Sciences. 81:7564-7568.) respecting the modulation of T cells may be erroneous. There, it was demonstrated that planar membranes containing purified MHC loaded with antigen fused to glass cover slips elicited IL-2 production by T cells through the interaction of the T cell with the MHC:antigen complex. It was also shown that the same complex when formed in unilamellar vesicles (i.e., liposomes) elicited no response. Contrary to such teaching, we have found that liposome vesicles containing MHC:antigen complexes can in fact elicit strong response when combined with accessory molecules such as LFA-1, and other molecules such as co-stimulatory and adhesion molecules. We based our theory that liposomes could function without use of a planar array on the observation (by the same study cited immediately above) that crude membrane preparations of cellular material from which the MHC was purified were effective in eliciting T cell responses in both planar and vesicular forms. Subsequently, we have discovered that xe2x80x9cextraneousxe2x80x9d matter existing in cell extracts that might be hypothesized to impart functionality to vesicular forms of lipid bilayers (as opposed to unilamellar liposomes alone) are not important to T cell binding and response. Rather, T cell binding and response is possible using vesicular forms of liposomes containing specific molecules applied in combination with lipsomes (e.g., accessory molecules, co-stimulatory molecules, and adhesion molecules).
Prior research has also been inconclusive respecting the use of MHC molecules. For example, it has been shown (Buus, S. Cell. 47:1071-1077.) that a particular antigenic peptide binds solely to the alpha chain of the class II MHC IAd molecule while other investigations have shown that binding interactions between T cell receptors and MHC:antigen ternary complexes use whole MHC, not just single chains of the MHC, to determine peptide sequence motifs. Exactly how much of a MHC:antigen complex must be presented is not absolutely known and may vary with T cell specificity. We have directed our invention to the use of either whole MHC molecules or those parts of the xcex1 and xcex2 subunits of Class I and Class II MHC necessary for forming antigen binding cleft regions in the binding of antigen peptides.
The current invention""s use of co-stimulatory, adhesion and other accessory molecules in a xe2x80x9cfree floatingxe2x80x9d format also helps to both anchor and direct the interaction between MHC:antigen:accessory molecule and T cell receptors by providing a means by which T cells in the sample will be presented with a structure more similar to that found in the natural state. Specifically, the MHC:antigen:accessory molecule complexes in conjunction with other functional molecules are able to migrate in proper orientation in the lipid bilayer of the liposome because of the use of a unique combination of lipids and surfactant molecules, namely an optimal ratio of phosphotidylcholine and cholesterol respectively, included in the liposome matrix. These provide particular protein presentation characteristics and easy protein migration properties to the surface of the liposome structure so that the MHC:antigen complexes can easily migrate to T cell binding loci similar to xe2x80x9ccappingxe2x80x9d events seen in natural APCs. Moreover, as shown in the figures, the structure of our artificial APC liposomes allows for specific xe2x80x9ccappingxe2x80x9d of the liposomes on the surface of the T cells to which the liposomes are bound. Additionally, interaction between the T cell and artificial APC-associated molecules is further enhanced by the molecules being oriented in the lipid membrane such that their active sites are positioned facing outward on the APC. Without such orientation, the ratio of properly oriented molecules to improperly oriented molecules is around 50:50. This ratio is greatly increased using MHC, functional and accessory proteins that have attached thereto (either by fusion protein construction or by use of a linker) a cholera xcex2 toxin subunit moiety which is placed in relation to the active center of the protein of interest such that upon the xcex2 subunit being bound by GM-1 which is incorporated into the lipid layer of the artificial APC, the protein of interest will lay in the APC with the active site facing outward.
Additional versatility is available with the current invention in that the artificial APCs may incorporate irrelevant molecules to be used in conjunction with separate solid support-based capture moieties for capturing generic target motifs such as irrelevant molecules. Because of the capacity for the functional molecules to migrate in the liposome, the irrelevant molecules may be nonspecifically directed away from the binding position of the T cells thus avoiding steric hindrances. Additionally, the system avoids a need for manufacturing specialized solid phase capture substrates for each antigen-specific complex.
With regard to the capture of the APC by the solid phase component of the invention, we refer to target molecules used in the artificial APC for binding to capture molecules of the solid support as xe2x80x9cirrelevantxe2x80x9d molecules because they do not impact the APC:T cell interaction. Such a design further preserves the ability of the other molecules inserted into the liposome to move freely and accommodate any capping of the T cell""s activation related molecules.
It has been recognized that the number of receptors on a T cell is variable (Rothenberg, E. Science. 273:78-79.). It is also known that the number of TCRs and combination of co-stimulatory molecules and accessory molecules varies with the maturation of the T cell (Dubey, C. J Immunol. 157:3820-3289.). How many such receptors are needed in all situations to elicit a T cell response is unknown. Moreover, it is known that presence of a co-stimulatory signal decreases the number of receptors necessary to activate a T cell (Viola, A. and Lanzavecchia, A. Science. 273:104-106.). We have provided for the uncertainties presented by such data by providing a system that allows control over the number of MHC:antigen:accessory molecule complexes relative to other functional molecules such as co-stimulatory, and adhesion molecules. The binding and modulation of the T cell response at different stages of cell maturation may be xe2x80x9cfine tunedxe2x80x9d using our invention.
In another system, Nag et al. in U.S. Pat. No. 5,734,023 (the ""023 disclosure), disclosed MHC subunits which were complexed with antigenic peptides and xe2x80x9ceffectorxe2x80x9d molecules wherein such complexes were used to identify T cell populations that were associated with autoimmune diseases. The complexes were used to destroy and anergize such T cell populations from a patient""s blood cell population.
The effector molecules are described as such things as toxins, radiolabels, etc. which may be conjugated to the MHC or antigen portion of the complexes and which may effecutate the identification, removal, anergy, or death of such T cell populations. Such effector molecules are not related to the attractive binding interactions or T cell responses to effectuate a phenotype change in the cells. They are merely designed and intended to aid in the recognition and/or destruction of specific T cell populations. Additionally, the ""023 disclosure uses lipids in the construction of micelles which are designed for intravenous injection as therapeutics. The use of negatively charged acidic phospholipids (such as phosphatidylserine) and the lack of cholesterol or GM-1 and cholera toxin subunit in the design of such micelles differs from that of the current invention in substantial ways. For example, our invention uses neutrally charged phospholipids such as phosphotidylcholine (Pc). We have found that the design of our artificial APCs substantially increases stability because of the Pc and cholesterol in environments where IL-1 is present. IL-1 is known to interact with charged phospholipids and destabilize liposome structure. Likewise, in environments where TNF is present, the permeability of liposomes comprised of charged phospholipids (e.g., phosphotidylserine) is greatly affected. In the same manner, environments where RNase is present may also affect charged phospholipid liposome structures. We have avoided the disruptive effects caused by molecules that are often present in media from which T cells are isolated by designing artificial APCs using neutral phospholipids.
Additionally, the use of liposomes and the parameters associated with micelle construction that are disclosed in the ""023 disclosure are wholly associated only with the stability of MHC:antigen:effector molecule complexes in the in vivo circulatory environment. There is no relation inherent or otherwise to the current invention, nor is there insight disclosed as to liposome construction containing co-stimulatory and adhesion molecules or protein orientation mechanisms such as the binding of cholera toxin by GM-1, or fused or linked moieties to the MHC, functional or accessory proteins of interest. Further, the ""023 disclosure does not discuss use of its micell construction in the context of use of a MHC:antigen complex ex vivo where manipulation of T cell function and the binding attraction between T cells and MHC:antigen complexes with respect to the current invention is of import. Moreover, the current invention does not use the technology disclosed in the ""023 disclosure of single chain MHC in liposomes. In contrast, in a preferred embodiment, our invention uses either whole MHC molecules or those portions of the xcex1 and xcex2 subunits necessary to bind antigens and that may be designed to have substantially favorable liposome stabilizing characteristics as well as binding capabilities when in the presence of other functional molecules in the artificial APC as disclosed herein.
In yet another recent disclosure, Spack et al. in U.S. Pat. No. 5,750,356 (the ""356 disclosure) describe a method for monitoring T cell reactivity using a modified ELISPOT assay which detects various factors produced by the stimulation of T cells with numerous factors in the presence of natural antigen presenting cells. The current invention is distinguishable from the ""356 disclosed method in that the current invention uses artificial antigen presenting cells which have incorporated therein various accessory, co-stimulatory, adhesion, cytokine, and chemokine molecules that provide substantial effect in the binding and modulation of T cell responses. Additionally, in embodiments that require solid support binding, our APC includes irrelevant molecules. Moreover, in another embodiment, our invention includes mechanisms to properly orient proteins of interest in the lipid membrane.
In still another disclosure, Wilson et al. in U.S. Pat. No. 5,776,487 disclose a use of liposome structures for determining analyte in a test sample wherein the liposome contains only an analyte specific ligand and a haptenated component used to bind to a receptor moiety on a solid phase. This combination allows for capturing a test analyte onto a solid support for detection. Thus, it is vastly divergent from the concept of the current invention.
Our methods and artificial APCs are further distinguished from other recent disclosures. For example, Altman et al., in U.S. Pat. No. 5,635,363 (the ""363 disclosure), discuss a method for labeling T cells according to the specificity of their antigen receptor by preparing a xe2x80x9cstable multimeric complexxe2x80x9d comprised of four or more MHC molecules having a substantially homogenous bound peptide population. The multimeric antigen:MHC complex was said to form a stable structure that because of its xe2x80x9cstable multimericxe2x80x9d design, purportedly increases the affinity of a T cell receptor for its specific antigen thereby allowing for the labeling, identification and separation of T cells. Although such multimeric MHC compoents are known to bind T cells, they are not incorporated into liposome structures. Thus, the MHC complexes are unable to participate in capping type concentration. Moreover, the ""363 method does not use accessory, co-stimulatory, adhesion, or other molecules to assist T cell binding and/or activation or modulation.
The current invention is further distinguishable over prior disclosures in that our invention is based on the recognition that the valency of the liposome:MHC structure is multiple, and empirically determined. Moreover, we have provided for greater specificity in following APC:T cell interaction due to one embodiment of our invention wherein the antigen is labeled rather than the MHC component (e.g. a biotinylated antigen with a streptavidin molecule conjugated to a fluorochrome).
In light of the above noted distinctions, the disclosed artificial APC and use of a separate solid support containing a binding protein to bind irrelevant molecules on the artificial APC represents an especially notable improvement over prior art.
For example, in the above mentioned prior technologies the design of complexes are such that simultaneous binding and capping of the MHC:antigen and TCR/CD3/accessory molecules cannot occur. Capping is the phenomenon by which the T cell focuses the relevant molecules to the portion of the cell where binding has occurred, thus amplifying the binding, and subsequently the signaling of the event to the cell""s other components. The current invention provides a specifically designed lipid bilayer similar to that of a natural cell which allows protein molecules, such as the MHC:antigen complexes to float freely, thus enabling the complexes to conform to any capping events the T cell may undergo. The consequence is a greater ability of the current invention to bind to, stimulate, and modulate T cells on demand.
The present invention is directed to novel methods of isolating T cells specific for particular antigens of interest and modulating T cell function ex vivo which methods use, in various embodiments, flow cytometry and immunoaffinity chromatography. Additionally, the present invention is directed to artificial antigen presenting cells (artificial APCs) and methods of making artificial APCs. In a preferred embodiment, such artificial APCs are used to isolate, expand, and modulate antigen-specific T cells. Additionally, the present invention is directed to methods of treating conditions which would benefit from the modulation of T cell responses, for example, transplantation therapies, autoimmune disorders, allergies, cancers and viral infections or virtually any T cell mediated disease. The present invention is further directed to a T cell modulation column device as well as a kit for isolating and modulating antigen-specific T cell populations.
Artificial APCs and APC Content
In one aspect, artificial APCs are provided having a synthetic membrane-based vesicle, such as a liposome containing cholesterol and neutral phopholipids such as phosphotidylcholine, that functions as an APC having capacities equivalent to a natural APC to bind to and induce an antigen-specific T cell response. Such an artificial APC comprises multiples of homo- or heterogenous combinations of MHC:antigen complexes incorporated therein as well as other functional molecules including accessory molecules, co-stimulation molecules, adhesion molecules, and other immunomodulatory molecules such as cytokines, cytokine receptors, chemokines, and chemokine receptors. Additionally, these APCs may include a mechanism to properly orient these molecules of interest in the APC membrane.
In one embodiment, accessory molecules may be used to facilitate and stabilize the interaction between the antigen specific T cell and the MHC:antigen complex. In this embodiment, an example of an accessory molecule is LFA-1. Other accessory molecules include, but are not limited to CD11a/18, CD54(ICAM-1), CD106(VCAM), and CD49d/29(VLA-4), as well as antibodies to each of these molecule""s ligands.
In another embodiment, the artificial APC includes co-stimulatory molecules that function to stimulate or activate an antigen-specific T cell. One form of activation is cell proliferation. Suitable co-stimulatory molecules include, but are not limited to, B7-1, B7-2, CD5, CD9, CD2, CD40 and antibodies to their ligands. Preferably, such co-stimulatory molecules can be produced by recombinant methods. Co-stimulatory molecules can be used for a variety of purposes in addition to eliciting cell proliferation. For example, it is known that memory CD4+ T cells express B7-2 whereas naive CD4+ T cells do not. Neither type cell expresses B7-1 (Hakamada-Taguchi, R. European Journal of Immunology. 28:865-873.). Thus, the current invention may be used to selectively target memory T cells by incorporating anti-B7-2 into the artificial APC complex.
In another embodiment, the artificial APC also includes adhesion molecules to facilitate strong and selective binding between the artificial APC and antigen-specific T cells. Suitable adhesion molecules include, but are not limited to, proteins of the ICAM family, for example ICAM-1 and ICAM-2, GlyCAM-1, as well as CD34, anti-LFA-1, anti-CD44 and anti-beta7 antibodies, chemokines, and chemokine receptors such as CXCR4 and CCR5, and antibodies to Selectins L, E, and P. Such molecules are known to be important as homing molecules for cells destined for specific locations in vivo. For example, Alpha4beta7 and L-selectin have been proposed as gut and peripheral lymphnode homing molecules respectively. Alpha4beta7 is expressed mainly on memory T cells while L-selectin is expressed mainly on naxc3xafve T cells (Abitorabi, M. A. J Immunol. 156:3111-3117.). It is also known that endothelial selectins (E-selectin and P-selectin) are associated with the extravasasion of T cells into inflammatory sites in the skin (Tietz, W. J Immunol. 161:963-970.). In the current invention a beta7 binding molecule and gut addressin MAdCAM-1, or an anti-L-selectin antibody may be incorporated into the artificial APC to distinguish further the type of T cell binding to the MHC:antigen complex.
In another example, it is known that CD44, which binds hyaluronan, is involved in leukocyte extravasation. Anti-CD44 antibody will bind to CD44 and strip it from the leukocyte surface. In one embodiment of the invention, anti-CD44 is incorporated into an artificial APC for use in stripping CD44 from leukocytes as desired thereby helping to inhibit the extravasation of the cells into extracellular spaces once the treated cells are returned to the patient. In another embodiment the anti-CD44 can be infused into an immunomodulatory column where the leukocytes have been captured by artificial APCs for the same purpose.
In another embodiment, other functional molecules (i.e., modulation molecules) may be incorporated into the artificial APC to facilitate T cell modulation. Examples of such molecules which may be incorporated into the artificial APC include, but are not limited to, CD72, CD22, and CD58, or antibodies to their ligands, antibodies to cytokine or chemokine receptors or small molecules which mimic the actions of the various cytokines or neuropeptides. These modulation molecules may be used for example to modulate the phenotype of antigen-specific T cells.
In another embodiment of the invention, the artificial APCs may also comprise irrelevant molecules which are included for the purpose of providing a means to anchor the APC to a solid support or to carry a label. Such molecules are termed irrelevant because they do not interact with the binding, activation, or modulation of the T cells.
In still another embodiment, any of the aforementioned molecules of interest (i.e., MHC, functional, accessory, irrelevant) may be bound to a cholera toxin subunit moiety by either a linking moiety of by a recombinant construction of a fusion peptide wherein the toxin subunit is linked directly to the protein of interest. In such embodiment, the cholera toxin subunit is positioned with respect to the molecule of interest such that the active portion of the molecule of interest is available for contact with T cells while the toxin portion remains in the the nonpolar region of the lipid layer of the APC. In a preferred embodiment, the cholera toxin moiety remains in the APC""s interior by binding to GM-1 that is incorporated into the APC""s lipid interior.
In still another embodiment, the APC comprises phospholipids, cholesterol, and GM-1 molecules each present in an appropriate ratio to allow free migration of molecules of interest around the lipid layer. Phospholipids contemplated include neutrally charged phospholipids such as phosphotidylcholine and cholesterol. Additionally, the cholesterol provides a surfactant property allowing the phospholipid to carry the various molecules of interest (accessory, irrelevant, modulation, adhesion, and co-stimulatory) in a manner that aids the free mobility of such molecules within the liposome membrane layer without disruption of the membrane in ex vivo environments.
In still another embodiment, the APC comprises antigens wherein the antigens are presented by an MHC components for contact with and recognition by a T cell receptor. Such antigens may be selected from the group consisting of a peptide, a peptide derived from the recipient for graft versus host diseases, a cancer cell-derived peptide, a peptide derived from an allergen, a donor-derived peptide, a pathogen-derived molecule, a peptide derived by epitope mapping, a self-derived molecule, a self-derived molecule that has sequence identity with said pathogen-derived antigen, said sequence identity having a range selected from the group consisting of between 5 and 100%, 15 and 100%, 35 and 100%, and 50 and 100%.
In still another embodiment, the APC comprises labels wherein a label is associated with at least one of the group selected from the group consisting of a lipid bilayer of the liposome components, a lipid of the liposome, an antigen, an MHC molecule, a co-stimulatory molecule, an adhesion molecule, a cell modulation molecule, GM-1, cholera toxin xcex2 subunit, an irrelevant molecule, and an accessory molecule.
Artificial APC Formation
In a preferred embodiment, artificial APCs may be made by:
(a) obtaining MHC:antigen complexes of interest;
(b) combining said MHC:antigen complexes and accessory molecules such as ICAM-1, with an artificial lipid membrane comprising the aforementioned lipids, cholesterol, and GM-1 molecules to form membrane-associated MHC:antigen:accessory molecule complexes, (i.e., liposome:MHC:antigen:accessory molecule complexes); and
(c) combining said liposome:MHC:antigen:accessory molecule complexes resulting from step (b) with one or more types of functional molecules (i.e., other accessory molecules, co-stimulatory molecules, adhesion molecules, modulation molecules, and irrelevant molecules) to form an artificial APC comprising liposome:MHC:antigen:accessory molecule:functional molecule complex. Preferably, steps (b) and (c) are performed simultaneously. Additionally, in this embodiment example, as well as all others mentioned herein, each of these molecules incorporated may be prelinked to cholera toxin (as by fusion protein construction or linker moiety).
In one embodiment, the functional molecules are individually optional. In another embodiment the irrelevant molecules are optional.
In another preferred embodiment, the artificial APCs may be made by:
(a) obtaining a spheroid solid support of interest having affinity for non-polar regions of a phospholipid; and
(b) combining MHC:antigen complexes, accessory molecules such as LFA-1, and functional molecules (i.e., other accessory molecules, co-stimulatory molecules, modulation molecules, irrelevant molecules, and adhesion molecules) with the phospholipid, cholesterol, GM-1 components and solid support to form a solid support associated:membrane-bound:MHC:antigen:accessory molecule:functional molecule complexes (i.e., solid support:phospholipid:MHC:antigen:accessory molecule:functional molecule complex) wherein of the molecules of (b), none are covalently bound to the solid support except optionally the lipid component.
In this embodiment, the solid support is preferably a glass bead or magnetic bead. It is also preferred that the phospholipid be phosphotidylcholine. In one embodiment of this aspect, the functional molecules are individually optional. In another embodiment, the solid support is either a glass or magnetic bead and has a diameter of between 25 and 300 xcexcm. Additionally, another solid-support APC construct has only lipids, cholesterol and a capture moiety having affinity for capturing an irrelevant molecule that is located on a non-solid-support APC. In such construct, the lipid layer is generally a monolayer.
Artificial APC Methods of Use
In another embodiment, the present invention is directed to a method of isolating T cells specific for an antigen of interest using an artificial APC comprising:
(a) obtaining a biological sample containing T cells which are specific for an antigen of interest;
(b) preparing a liposome:MHC:antigen:accessory molecule functional molecule complex (i.e. artificial APC), wherein the antigen in said complex is said antigen of interest;
(c) contacting the biological sample obtained in step (a) with the artificial APC obtained in step (b) to form a artificial APC:T cell complex;
(d) removing said complex formed in step (c) from said biological sample; and
(e) separating T cells specific for said antigen of interest from said complex formed in step (c).
Optionally, such a method of isolating T cells specific for a particular antigen of interest may include the step of determining the quantity of such T cells complexed with the artificial APC, and/or may include the step of characterizing the functional phenotype of such T cells. Preferred biological samples containing T cells specific for an antigen of interest include bodily fluids such as blood, blood plasma, and cerebrospinal fluid. Other suitable biological samples include solid tissue, for example histological specimens.
In a preferred embodiment, the method uses FACS technology, and the antigen is labeled. Preferred labels include biotin, fluorochromes and radioactive labels. For example, one type of label which may be used is vancomycin (Rao et al., Science, Vol. 280:5364:708-11, 1998). In another embodiment of the method, also using FACS technology, the liposome may be labeled or the label may be noncovalently enclosed within the liposome matrix. If the label is within the liposome matrix, the label may be either enclosed within the liposome or incorporated within the lipids of the outer membrane of the liposome. In another embodiment, the irrelevant molecule, if present, may be labeled. In yet another embodiment, the complex of the artificial APC and T cell may be removed from the biological sample by capturing the complex via the irrelevant molecule on to a solid support. In such case, a solid support comprises an irrelevant molecule binding or capture molecule (e.g. anti-irrelevant molecule antibody) bound either directly to the solid support or noncovalently associated with a phospholipid bound to the support.
In another embodiment, the present invention provides an alternate method of isolating T cells specific for an antigen of interest. This alternate method comprises:
(a) contacting an artificial APC having a MHC:antigen:accessory molecule component of interest with a solid support to form a solid support:artificial APC (The liposome of the APC contains a binding molecule i.e., an xe2x80x9cirrelevantxe2x80x9d binding molecule. The capture molecule that captures the irrelevant molecule may be bound to said solid support via a linker or may be associate with a phospholipid layer on the solid support). In this embodiment, the antigen binding region of said MHC:antigen component is available for binding to a T cell receptor without steric hindrance because the MHC:antigen component is free to move within the liposome membrane of the APC while the irrelevant binding protein allows the APC to be anchored to the solid support;
(b) contacting said solid support:artificial APC with a biological sample containing T cells specific for an antigen of interest to form a solid support:artificial APC:T cell complex;
(c) removing said solid support:artificial APC:T cell complex from said biological sample; and
(d) separating the T cells specific for said antigen of interest from said complex.
In a further aspect of the present invention, kits for the isolation of T cells specific for an antigen of interest are provided. In one embodiment, the kits comprise:
(a) APCs and solid supports such that there is included APCs having MHC (and other functional molecule where desired) complexes; and
(b) materials well known to those knowledgeable in the art which facilitate the completion of the isolation of an antigen-specific T cell population including, but not limited to, buffers, culture medium, (included in buffers may be cytokines, antibodies to various transmembrane or soluble molecules, chemokines, neuropeptides, or steroids), (included in culture medium may be cytokines, antibodies to various transmembrane or soluble molecules, chemokines, neuropeptides, or steroids), antigens, MHC molecules, accessory molecules, co-stimulatory molecules, modulatory molecules and adhesion molecules.
In another kit embodiment, the kit may comprise a solid support having a means to capture an irrelevant molecule located in an artificial APC, and an artificial APC constructed as described above. In another embodiment, the kit may comprise virtual artificial APCs or solid supports comprising a lipid layer.
In another preferred embodiment, the invention includes an antigen-specific T cell isolation and modulation column device. In this embodiment, the device comprises compartments that may be isolated from one another having entrance and exit flow ports between said compartments and between the compartments and external apparatuses. Any of the compartments of such column device may further comprise solid supports capable of binding irrelevant molecules of artificial APCs or solid supports that function directly as artificial APCs as described above. Moreover, such a device may be used in connection with soluble immunomodulatory molecules that are neither bound to a solid support or incorporated into an artificial APC. Examples of such molecules include cytokines, chemokines and hormones.
In another such example, if leukopheresis is being performed with the intention of reintroducing the cells back into the patient""s body, soluble factors may be introduced into a column device to induce production of IL2 in naxc3xafve T cells, IL2 being necessary for T cell growth. Likewise, IL4 or soluble IL4 receptor antibody may be introduced into an immunomodulatory column to enhance the Th2 phenotype in specific T cells of interest.
The invention further comprises a method of modulating T cell responses (i.e., altering a T cell""s phenotype). In such embodiment, methods of regulating or modifying T cell responses ex vivo, such as in a column device of the invention, are provided comprising the steps of isolating T cells which are specific for an antigen of interest and combining said isolated T cells with an artificial antigen presenting cell. The aforementioned steps may be performed simultaneously or separately as by addressing antigen-specific T cells from one compartment to another after first capturing the T cell followed by introduction of an artificial APC. Preferably, the T cells specific for an antigen of interest are isolated using the T cell isolation methods described above.
The modulation of T cell response may comprise changing, in whole or in part, the functional pattern of cytokine receptor expression, cytokine production, chemokine production, and/or chemokine receptor expression by the isolated T cells specific for a given antigen. For example, a T cell may be stimulated to shift its phenotype from a Th0 to a Th1. In another example, a T cell may be stimulated to shift its phenotype from a Th1response to a Th2 response. In yet another example, a T cell may be stimulated to shift from any other phenotype to a Th3 phenotype. Amongst the many possible means to induce modulation of a T cell response for the purpose of increasing its Th2 response and/or decrease its Th1 response, preferably the artificial APC used in such method expresses the co-stimulatory molecule B7-2.
In another embodiment, the modulation of T cell response may comprise changing, in whole or in part, the functional pattern of cytokine production by said isolated T cells from a Th2 response to a Th1 response. Preferably, to modify a T cell response to induce it to increase its Th1 response and/or decrease its Th2 response, the artificial APC used in such method expresses the co-stimulatory molecule B7-1.
In another example of T cell modulation, it is known that ST2L expression is Th2-type specific. In the current invention, ST2L may be included in the APC in order to identify, isolate and extract antigen-specific T cells of the Th2 phenotype and/or enrich T cell population for antigen-specific Th1 phenotype in the treatment of autoimmune disease.
In another example, OX40 ligand is known to induce a Th2-like phenotype in naxc3xafve T cells (Flynn, S. Journal of Experimental Medicine. 188:2:297-304.). In the current invention, OX40 may be incorporated into and artificial APC to selectively induce a Th2 phenotype.
In another example, CD30 is known to have association with asthma (Spinozzi, F. Mol Med. 1:7:821-826.). In the current invention, CD30 may be incorporated into artificial APCs to identify, isolate and remove antigen-specific T cells of the Th2 phenotype or to augment T cell response away from harmful T helper cells in the treatment of allergic conditions.
In yet another embodiment, the modulation of T cell response may comprise inducing anergy and/or apoptosis. Since it is known that the same cell need not present both the specific antigen and the co-stimulatory molecule for T cell activation (Ding, L and Shevach, E. M. European Journal of Immunology. 24:4:859-866.), our system is applicable to situations where the artificial APC does not express a co-stimulatory molecule, or contains another effector molecule, e.g. Fas ligand, to induce anergy. Thus, the artificial APC used in such method would not express a co-stimulatory molecule but may alternatively express Fas ligand.
In yet another embodiment, the modulation of T cell response may comprise inducing T cell proliferation in general, without regard to modifying Th1 or Th2 response, and without regard for inducing anergy. Preferably, this is accomplished by an artificial APC that expresses an anti-CD28 antibody.
In yet another preferred embodiment, the present invention provides methods of treating a condition in a subject who would be benefited by modulating the functional pattern of active factors expressed by a T cell. Such method of treatment could include in addition to the use of artificial APCs, the use of a column device described herein. For example, in such a treatment regimen, production of cytokines by a T cell may be modified in certain antigen-specific T cells to increase Th2 response and/or decrease Th1 response. In such a method, a subject""s T cells that are specific for an antigen capable of triggering a Th1 response are isolated by contacting said cells with an APC having an MHC:antigen complex containing an appropriate antigen. By also including the co-stimulatory molecule B7-2 on the APC, the T cells may be directed to modify their response and cytokine production to increase a Th2 response. Conditions which would be benefited by altering the functional pattern of response toward a Th2 response include, for example, autoimmune diseases such as type 1 diabetes mellitus, multiple sclerosis, rheumatoid arthritis, dermatomyositis, juvenile rheumatoid arthritis and uveitis.
In another example of a method of treatment, it is known that the cross-linking of the CD40 ligand by means of antibodies induces cell proliferation and IL4 production (Blotta, M. H. J Immunol. 156:3133-3140.). Additionally, it is known that blockade of CD40/CD40 ligand pathway induces tolerance in murine contact hypersensitivity (Tang, A. European Journal of immunology. 27:3143-3150.). In the current invention, CD40 or anti-CD40 ligand antibody may be incorporated into an artificial APC to induce T cell modulation toward production of IL4 and/or tolerance to alleviate inflammatory autoimmune disorders.
In yet another example of a method of treatment, a subject may be benefited by altering the functional pattern of cytokine production by certain antigen-specific T cells to increase Th1 response and/or decrease Th2 response. Such methods comprise isolating a subject""s T cells that are specific for an antigen capable of triggering a Th2 response by contacting said cells with an APC containing an MHC:antigen complex having an appropriate antigen, wherein said artificial APC also expresses the co-stimulatory molecule B7-1. Conditions which would be benefited by altering the functional pattern of cytokine production to increase Th1 response and/or decrease Th2 response include, for example, allergy, for example allergy to dust, animal skin bypass products, vegetables, fruits, pollen and chemicals. Other conditions which may be benefited include cancers and some types of infections (e.g., viral, protozoan, fungal and bacterial).
In another example of a method of treatment, it is known that anti-B7-1 antibody will reduce the incidence of EAE, an animal model of multiple sclerosis. Anti-B7-2 antibody is known to increase the severity of EAE, while co-treatment with anti-IL4 antibody will prevent disease amelioration. In the current invention, more than one level of control with respect to this disease is possible. For example, artificial APCs may be generated that express anti-B7-1 antibody and/or IL4 to elicit T cell response favorable to treating the disease.
In another example, a regimen may be developed for treating melanoma by screening for T cell responses to epitopes derived from MAGE-1, MAGE-3, MART-1/melan-A, gp100, tyrosinase, gp75, gp15, CDK4 and beta-catenin, all of which are known to be associated with the disease. T cells having specificity for these molecules may be activated and modulated in various ways. For example, T cells that are specific for a unique cancer related antigen can act to cause destruction of the cancerous cells may be proliferated and infused into a patient.
In another example of a method of treatment, artificial APCs may be designed to augment antigen-specific T cell response away from the harmful helper type T cells or used to deplete offending T cells in the treatment of multiple sclerosis. Such depletion or modulation of the T cells may be carried out in combination with the infusion into a column device of either Fas, Fas Ligand, anti-Fas or anti-Fas ligand antibody. Additionally, artificial APCs may be designed to incorporate SLAM reactive molecules into the liposome complex which functions to induce a suppressive phenotype.
In another treatment example, SLAM reactive molecules incorporated in an artificial APC may be used to treat Th2-mediated autoimmune diseases by modulating the T cell response to shift from a Th2 profile to a Th0/Th1 profile while at the same time inducing IL-2 independent, co-stimulation independent proliferation.
In yet another treatment example, cartilage degradation that is associated with rheumatoid arthritis can be prevented by use of IL-10 and IL-4 in an artificial APC or by infusion of the soluble molecules into a column device to modulate antigen-specific T cells away from activated Th1 state.
In yet another treatment example, IL-12 can be used such as by infusion into a column device or incorporation in an artificial APC to induce Th1 type inflammatory response to help treat Th2 mediated autoimmune diseases.
In another example of a treatment method, T cell mediated milk intolerance may be treated by isolation and depletion of T cells specific for the allergen which is incorporated as an MHC bound antigen on an artificial APC.
In still another example of using the invention to treat cancerous conditions, T cells specific for the ras peptide, or variants thereof, may be stimulated by response to an artificial APC containing the ras peptide in the treatment of cancerous cells expressing the ras mutation.
In another example of the advance of the current invention over that of currently applied art, instead of observing the effects of specific molecules on T cell response in vivo, the current invention allows one to follow T cell modulation ex vivo. For example, previous studies (Gaur, A. J of Neuroimmunol. 74:149-158.) showed that using I.V. injection of non-encephalitogenic APL91 peptide of MBP ameliroates disease by shifing cytokines from Th1 to Th2 phenotype. The same type of injections using encephalitogenic superagonist APL A97 ameliorates disease by causing deletion of specific T cells. The problem arises that understanding the bioavailability of the injected complexes is difficult at best while the ex vivo methodology of the current invention allows one to follow the specific actions of the peptides when used either in an artificial APC or in soluble form in a column device.
In another situation, prior studies have indicated that in vivo application of peptides to treat autoimmune disease states is related to epitope spreading resulting in relapsing episodes of disease (Lehmann, P. V. Nature. 358:6382:155-157.),(McRae, B. L. Journal of Experimental Medicine. 182:75-85.). The ex vivo application of the current invention is preferred because specific peptides can be used to isolate and identify antigen-specific T cells without exposing a patient to the danger of epitope spreading that is associated with relapses of certain autoimmune diseases.
In a further preferred embodiment of the invention, a method of identifying T cells that express MHC epitopes important to graft versus host rejection in transplantation therapy is provided. In this embodiment, such MHCs are identified followed by their incorporation into the artificial APC. Such APCs may be used to capture and deplete the recipient""s T cells having specificity for such epitopes so as to allow a favorable modulation of the recipient""s immune response to the graft. More specifically, peptides derived from the MHC of a recipient are bound to the MHC of a donor that are incorporated into artificial APCs. Such APCs are used in combination with tolerogenic stimuli also on the APC or infused into the immunomodulatory column. The donor""s T cells are then screened to bind reactive T cells that can then be discarded. In a preferred embodiment, the column device of the invention can be used to carry out such immunoleukophoresis.
In another embodiment, the method contemplates treatment of an individual to cause a favorable immune modulation to an allograft comprising:
(a) predicting a donor""s MHC to which a recipient may react;
(b) testing the predicted MHC epitopes with a recipient""s T cells to identify antigenic epitopes;
(c) using identified epitopes in an artificial APC to deplete the recipient""s antigen-specific (i.e., donor-specific) T cells while additionally desensitizing the recipient to the epitope by contacting the recipient (as by feeding or nasal injection) with increasing doses of the donor-specific antigen.
In still another preferred embodiment, the invention provides a method of identifying an individuals""MHC epitopes that are of import in immunologic responses to pathogenic agents. In this embodiment, an individual""s MHC is screened for epitopes that have appreciable sequence or molecular structure recognition with pathogen-derived molecules. The identified MHC epitopes can be used to elicit immunity that may be employed directly as a vaccine against such MHC epitopes that have sequence recognition with pathogen-derived peptides. For example, such a vaccine may be used to reduce natural APCs that express MHC molecules associated with autoimmune diseases including, but not limited to, multiple sclerosis, rheumatoid arthritis, and diabetes. In another preferred embodiment, the identified MHC epitopes can be incorporated into a liposome structure as a co-stimulatory molecule to enhance the effect of artifical APCs that express other disease related antigens.
In yet another preferred embodiment, antigenic moieties of the pathogen which have or are likely to have MHC mimics may be used in artificial APC MHC:antigen complexes to isolate T cells specific for such pathogen-derived antigenic motifs or mimics thereof for the production of a T cell vaccine. Such a vaccine may be used to directly fight progression of an infection or disease caused by a pathogen or that is the result of a pathogen-derived antigen induced autoimmune associated inflammatory response. Usually a self-derived molecule that mimics a pathogen-derived antigen comprises a polypeptide. As used herein, such a mimic has an amino acid sequence identity with said pathogen-derived antigen to an extent necessary for the MHC to bind to the mimic. The range of sequence identity may be anywhere between 5 and 100% depending upon which amino acids in a peptide sequence elicits either recognition by the MHC and/or stimulation of a T cell response. Generally, the range is between 5 and 100%, usually, the range is between of a range of 15 and 100%, preferably the range is between 35 and 100%, and most preferably the range is between 50 and 100%.
In still other embodiments, the invention provides a means to address other immunologic conditions relevant to T cell response. For example, apoptosis of T cells induced by MHC molecules through the CD95/CD95 ligand pathway can be controlled by incorporating anti CD95 into an artificial APC and modulating antigen-specific T cells.
In another application, dendritic cells important to immune response may be manipulated ex vivo in the same fashion as T cells in the many examples provided above.